Study of CALR, MPL, and c-kit Gene Mutations in Thai Patients with JAK2 V617F Negative Myeloproliferative Neoplasms.

OBJECTIVE: The aim of this study to determine the prevalence of CALR, MPL and c-kit gene mutations in JAK2 V617F negative-MPN patients. METHODS: The retrospective study of CALR, MPL and c-kit mutations were analyzed in 113 samples collected from March 2010 to May 2017 and identified as JAK2 V617F-negative MPN Thai patients. The samples were analysis by gel electrophoresis and direct sequencing. RESULTS: 28.3% of JAK2 V617F-negative MPN patients showed CALR gene mutations. Within the MPN patients with CALR mutation, 46.9% were classified as essential thrombocythemia (ET) and 20.9% were classified as primary myelofibrosis (PMF). Previous studies classified CALR mutations into three types using negatively charged amino acid stretches at the C-terminal domain. Type 1-like mutations were observed in 12 of 49 (24.5%) ET patients and type 2-like mutations were observed in 10 of 49 (20.4%) patients. In addition, 8 of 43 (18.6%) PMF patients showed type 1-like mutations and 1 of 43 (2.3%) showed type 2-like CALR mutation. Interestingly, platelet counts were higher in patients with CALR gene mutation than in patients without CALR gene mutation. MPL mutations (W515K and W515L) were identified in 2 of 109 (1.8%) MPN patients; the MPL mutations were only found in ET patients, which was consistent with previous studies. We did not detect exon 17 c-kit mutation in JAK2-negative MPN patients but detected intronic single nucleotide polymorphisms at c.74,978 and c.75,255 in these samples. Approximately 66% of patients did not have mutations in CALR and MPL genes, in addition to lacking JAK2 gene mutation, and these cases are classified as triple-mutations. CONCLUSION: Our results showed that 66% of cases were triple-negative mutation MPN because they lacked mutations in JAK2, CALR and MPL genes. The frequencies of CALR and MPL mutation in this study are similar to other CALR and MPL patient data.

Intracellular localization and cytotoxicity of Bacillus thuringiensis Vip3Aa against Spodoptera frugiperda (Sf9) cells.

Vip3Aa protein is produced by Bacillus thuringiensis during vegetative growth and displays high toxicity against a wide range of lepidopteran insect larvae such as Spodoptera exigua and Spodoptera frugiperda, both important insect pests worldwide. Vip3Aa protein is synthesized as a protoxin (proVip3Aa) and becomes activated by digestion with either trypsin or insect gut proteases. The activated Vip3Aa protein (actVip3Aa) binds to a specific receptor in the brush border epithelial midgut cells, causing cell death via apoptosis, possibly induced by its pore-forming activity. Here we investigated the actVip3Aa intracellular localization to explain the molecular mechanism leading to the cytotoxicity of Vip3Aa toxin. The Spodoptera frugiperda (Sf9) cell line was incubated with fluorescently labeled Vip3Aa, namely Alexa488-actVip3Aa, and the intracellular localization was analyzed through a laser scanning confocal microscope. The Alexa488-actVip3Aa was internalized into the Sf9 cells. Immunofluorescence detection demonstrated that Alexa488-actVip3Aa did not colocalize with early endosomes which is usually implicated in clathrin-mediated endocytosis, suggesting that the actVip3Aa does not use clathrin-dependent endocytosis to transport into the cytosol. Intracellular visualization also shows that actVip3Aa does not directly target to mitochondria upon entry into the cytosol. Following internalization, actVip3Aa causes cell division disruption that subsequently could trigger cell death via apoptosis.